Commercial practices have resulted in the production of pollutants that can contaminate the environment. For instance, modem agricultural practices rely heavily on the use of herbicides to control weed populations. S-triazine (i.e., symetric triazine) herbicides, primarily atrazine and simazine, are widely used herbicides for selective control of broadleaf weeds and some grasses in a variety of crops. Since atrazine and other s-triazine herbicides biodegrade relatively slowly in soils, label directions for the use of atrazine restrict the types of crops that can be planted to prevent carryover problems in the next growing season. For example, alfalfa and soybeans are susceptible to atrazine concentrations in soil ranging from 0.09 mg/Kg to 0.53 mg/Kg, depending on the concentration of soil organic matter.
Numerous studies on the environmental fate of atrazine have shown that atrazine is a moderately persistant compound that is transformed to CO2 very slowly, if at all, under aerobic or anaerobic conditions. It has a water solubility of 33 mg/l at 27xc2x0 C. Its half-life (i.e., time required for half of the original concentration to dissipate) can vary from about 4 weeks to about 57 weeks when present at a low concentration (i.e., less than about 2 parts per million (ppm)) in soil. High concentrations of atrazine, such as those occurring in spill sites, have been reported to dissipate even more slowly.
As a result of its widespread use, atrazine is sometimes detected in water in concentrations exceeding the maximum contaminant level (MCL) of 3 xcexcg/l (i.e., 3 parts per billion (ppb)), a regulatory level that took effect in 1992. Point source spills of atrazine have resulted in levels as high as 25 ppb in some wells. Levels of up to 40,000 mg/l (i.e., 40,000 ppm) atrazine have been found in the soil at spill sites more than ten years after the spill incident. Point source spills and subsequent runoff can result in the presence of atrazine in surface, subsurface, and ground water.
Atrazine removal from the soil environment can occur by several different mechanisms. At typical soil pH, atrazine is only very slowly chemically hydrolyzed (half life of 200 days) to produce hydroxyatrazine. A more significant degradation mechanism for atrazine in soils is microbial metabolism. Microbial degradation of atrazine has been demonstrated to occur via dealkylation, deamination, or dechlorination reactions.
For decontamination purposes, the most efficient method of transforming a contaminant into a less-harmful end product is by biostimulation or bioaugmentation (Liu et al. (1993) Trends Biotechnol., 11 344-352). Biostimulation involves supplementing the contaminated soils to change the physical state of the contaminant, thereby converting it to a bioavailable form, or supplying a nutritional supplement or co-substrate to increase the population of indigenous bacteria capable of catabolizing the contaminant. Bioaugmentation involves inoculating soils with a non-indigenous microorganism capable of catabolizing the contaminant.
The ability of introduced live cultures of atrazine-degrading bacteria to increase biodegradation has been investigated in laboratory studies. In studies done with non-sterile soil, the success of bioaugmentation was inversely related to population levels of indigenous atrazine-degrading microorganisms (Radosevich et. al., (1996) Biodeg., 7, 137-149; Struthers et al., (1998) Appl. Environ. Microbiol., 64, 3368-3375; and Kontchou et al., (1993) Proceedings of the 9th Symposium on Pesticide Chemistry, Piacenza Italy. p. 533-536. Istituto di Chimica Agraria et Ambientale, Universita Cattolica del Sacro Cuore). In sterile soils devoid of indigenous atrazine degrading bacteria, it has been reported that atrazine concentration was reduced 70% (from 20 ppm to 6 ppm) in 30 days (Fadullon et al., (1998) Environ. Sci. Health, B33, 37-49), or eliminated from 15 ppm in 5 days (Wenk et al, (1998) Appl. Mibrobiol. Biotechnol., 49, 624-630).
In view of the occasional prevalence of compounds, for instance herbicides, in the environment at levels above regulatory standards, and the long periods of time that can be required to allow natural degradation to occur, there is a need in the art for rapid methods to remediate, e.g., remove, pollutants present in the environment. The present invention represents an advance in the art of remediating compounds, for instance pollutants, in the environment. Typically, when a population of microbes expressing an enzyme activity of interest is exposed to conditions that result in 100% killing of the population, there is generally a substantial decrease in the amount of enzymatic activity retained by the cells when compared to the cells before killing. As described herein, when a population of microbes containing a hydrolase were incubated in a phosphate buffer and exposed to conditions that result in 100% killing, there was an unexpected high degree of hydrolase activity retained by the microbes when compared to the microbes before killing. When Na2B4O7xe2x80x94HCl was used as a buffer instead of phosphate, the level of hydrolase activity retained by the microbes compared to the microbes before killing was unexpectedly increased to an even greater degree than observed when the phosphate buffer was used. Also unexpected was the long term stability of the killed cells. For instance, after storage at room temperature for about seven months, killed microbes retained about 50% of enzyme activity of the enzyme activity that was present in the microbes before killing.
The present invention provides a method for remediating a compound in a sample. The method includes providing at least one killed microbe that contains a polynucleotide including a coding region encoding a polypeptide, for instance a hydrolase, that degrades a compound. The coding region can be an exogenous coding region. The microbe can be, for instance, E. coli or Pseudomonas aeruginosa. 
The method also includes contacting the sample that contains the compound with the at least one microbe under conditions effective to decrease the concentration of the compound in the sample relative to the concentration of the compound in a sample not contacted with the at least one microbe. The method can also include measuring the concentration of the compound in the sample after contacting the sample with the at least one microbe. The compound can be detoxified. The microbe can be killed with a cross-linking agent, for instance, glutaraldehyde, formalin, or iodine.
A sample that can be used in the methods of the present invention can include soil, water, or a combination thereof. The compound to be degraded can be at least one s-triazine, including for instance atrazine, desethylatrazine, deisopropylatrazine, desethylhydroxyatrazine, desisopropylhydroxyatrazine, desethyldesisopropylatrazine, simazine, terbuthylazine, melamine, ammelide, ammeline, prometryn, ametryn, propazine, cyanuric acid, terbutryn, cyanazine, propazine, simatone, and cyromazine.
The complement of the nucleotide sequence of a coding region useful in the present invention can include those that hybridize to the nucleotide sequence set forth at nucleotides 236 to 1660 of SEQ ID NO:3 in a solution containing 250 mM Na2HPO4, pH 7.4, 2 ml/liter 0.5 M EDTA, pH 8.0, and 10 grams/liter bovine serum albumin at 65xc2x0 C. for at least 4 hours, followed by three washes for twenty minutes each at 65xc2x0 C. in a solution containing 2xc3x97SSC and 0.1% SDS. The nucleotide sequence of the coding region can be nucleotides 236 to 1660 of SEQ ID NO:3. The amino acid sequence of the polypeptide can be the amino acid sequence of SEQ ID NO:4, or an active analog or active fragment thereof.
The invention also provides a method for degrading an s-triazine in a sample, including providing at least one cross-linked microbe that contains a polynucleotide including a coding region encoding a hydrolase that degrades an s-triazine. The method also includes contacting a sample that includes the compound with the microbe under conditions effective to decrease the concentration of the compound in the sample relative to the concentration of the compound in a sample not contacted with the at least one microbe. The microbe can be a prokaryote, including, for instance, E. coli and P. aeruginosa. Less than about 40% of individual microbes can be cross-linked to each other.
In another aspect, the invention also provides at least one cross-linked microbe containing a polynucleotide that includes a coding region encoding a polypeptide that degrades at least one s-triazine.
Definitions
xe2x80x9cMicrobexe2x80x9d and xe2x80x9cmicro-organismxe2x80x9d are used interchangeably herein and refer to a single-cell eukaryotic or prokaryotic organism. A microbe is xe2x80x9cisolatedxe2x80x9d when it has been removed from its natural environment and can be grown as a pure culture. An individual microbe is a microbe that is not cross-linked to another microbe.
xe2x80x9cBioremediationxe2x80x9d and xe2x80x9cremediationxe2x80x9d as used herein refer to decreasing the concentration of at least one compound in a sample. A sample can include, for instance, soil, a liquid, or both. The sample can be remediated while present in the environment, or remediated before being introduced to the environment. The concentration of a compound can be decreased by degrading the compound.
Water as used herein includes surface water, subsurface water, and ground water. xe2x80x9cSurface waterxe2x80x9d is water that is standing (e.g., a puddle) or moving (e.g., a stream) above ground level. xe2x80x9cSubsurface waterxe2x80x9d is water present in soil and above the ground water. Subsurface water includes water that entered the soil as rain and water that originated from, for instance, a nearby waterway. xe2x80x9cGround waterxe2x80x9d is water that is located below the subsurface water and often supplies wells and springs.
A xe2x80x9ccompoundxe2x80x9d as used herein refers to a molecule that is not typically in the environment, for instance a pollutant or a contaminant. A compound can be toxic to a plant or an animal.
xe2x80x9cS-triazinesxe2x80x9d and xe2x80x9cs-triazine containing compoundsxe2x80x9d are used interchangeably and refer to a type of compound. Examples of s-triazines include, for example, atrazine (2-chloro-4-ethlyamino-6-isopropylamino-1,3,5-s-triazine), desethylatrazine (2-chloro-4-amino-6-isopropylamino-s-triazine), deisopropylatrazine (2-chloro-4-ethylamino-6-amino-s-triazine), desethylhydroxyatrazine (2-hydroxy-4-amino-6-isopropylamino-s-triazine), desisopropylhydroxyatrazine (2-hydroxy-4-amino-6-isopropylamino-s-triazine), desethyldesisopropylatrazine (2-chloro-4,6-diamino-s-triazine), simazine (2-chloro-4,6-diethylamino-s-triazine), terbuthylazine (2-chloro-4-ethylamino-6-terbutylamino-s-triazine), melamine (2,4,6-triamino-s-triazine), ammelide (2,4-dihydroxy-6-amino-s-triazine), ammeline (2-hydroxy-4,6,-diamino-s-triazine), prometrym (N,Nxe2x80x2-bis(1-methylethyl)-6-(methylthio)-1,3,5-triazine-2,4 diamine), ametryn (N-ethyl-Nxe2x80x2-(1-methylethyl)-6-(methylthio)-1,3,5-triazine-2,4 diamine), propazine (6-chloro-N,Nxe2x80x2-bis(1-methylethyl)-1,3,5-triazine-2,4-diamine), cyanuric acid (trihydroxy-1,3,5-triazine), terbutryn (N-(1,1-dimethylethyl)-Nxe2x80x2-ethyl-6-(methylthio)-1,3,5-triazine-2,4-diamine), cyanazine (2-((4-chloro-6-(ethylamino)-1,3,5-triazine-2-yl)amino)-2-methylpropionitrile), propazine (6-chloro-N,Nxe2x80x2-bis(1-methylethyl)-1,3,5-triazine-2,4-diamine), simatone (methoxy-4,6-bis(ethylamino)-s-triazine), and cyromazine (2-Cyclopropylamino-4,6-diamino-s-triazine).
xe2x80x9cDegradationxe2x80x9d of a compound includes, for instance, removing or otherwise changing at least a portion of the compound. Degradation of a toxic compound can result in a compound having increased toxicity relative to the undegraded compound, a compound having about the same toxicity as the undegraded compound, or a compound having lower toxicity relative to the undegraded compound. A degraded compound having lower toxicity to a plant and/or an animal relative to the undegraded compound is referred to herein as detoxified.
xe2x80x9cKilledxe2x80x9d as used herein refers to a microbe that has been rendered incapable of reproducing and does not respire.
xe2x80x9cCross-linking agentxe2x80x9d as used herein refers to a chemical that, when exposed to a sample containing molecules, causes the formation of bonds between the molecules. The bonds can be, for instance, covalent, ionic, or hydrogen, preferably covalent. Exposure of a microbe to a cross-linking agent can result in the cross-linking of molecules within the microbe, and optionally the cross-linking of microbes. Cross-linking agents include agents that catalyze cross-linking but are not included in the resulting cross-linked molecule as well as agents that are included in the resulting cross-linked molecule.
xe2x80x9cHydrolasexe2x80x9d as used herein is a polypeptide that catalyzes hydrolysis, i.e., a chemical reaction in which water reacts with another molecule to form two or more new molecules. This involves the splitting of the molecule hydrolyzed. Non-limiting examples of hydrolases include phosphotriesterase, chlorohydrolases (for instance atrazine chlorohydrolase), nitrilases (for instance aliphatic nitrilase), and xcex2-galactosidase.
xe2x80x9cPolynucleotidexe2x80x9d as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides, and includes both double- and single-stranded DNA and RNA. A polynucleotide may include both coding and non-coding regions, and can be obtained directly from a natural source (e.g., a microbe), or can be prepared with the aid of recombinant, enzymatic, or chemical techniques. A polynucleotide can be linear or circular in topology. A polynucleotide can be, for example, a portion of a vector, such as an expression or cloning vector, or a fragment.
xe2x80x9cPolypeptidexe2x80x9d as used herein refers to a polymer of amino acids and does not refer to a specific length of a polymer of amino acids. Thus, for example, the terms peptide, oligopeptide, protein, and enzyme are included within the definition of polypeptide. This term also includes post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like.
xe2x80x9cCoding regionxe2x80x9d refers to a polynucleotide that encodes a polypeptide, usually via mRNA, when placed under the control of appropriate regulatory sequences. The boundaries of the coding region are generally determined by a translation start codon at its 5xe2x80x2 end and a translation stop codon at its 3xe2x80x2 end. xe2x80x9cExogenous coding regionxe2x80x9d refers to a foreign coding region, i.e., a coding region that is not normally present in a microbe, or a coding region that is normally present in a microbe but is operably linked to a regulatory region to which it is not normally operably linked.
xe2x80x9cRegulatory regionxe2x80x9d refers to a polynucleotide that regulates expression of a coding region to which a regulatory region is operably linked. Non-limiting examples of regulatory regions include promoters, enhancers, transcription initiation sites, translation start sites, translation stop sites, and terminators.
xe2x80x9cOperably linkedxe2x80x9d refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A regulatory element is xe2x80x9coperably linkedxe2x80x9d to a coding region when it is joined in such a way that expression of the coding region is achieved under conditions compatible with the regulatory region.
The term xe2x80x9ccomplementxe2x80x9d and xe2x80x9ccomplementaryxe2x80x9d as used herein, refers to the ability of two single stranded polynucleotides to base pair with each other, where an adenine on one polynucleotide will base pair to a thymine on a second polynucleotide and a cytosine on one polynucleotide will base pair to a guanine on a second polynucleotide. Two polynucleotides are complementary to each other when a nucleotide sequence in one polynucleotide can base pair with a nucleotide sequence in a second polynucleotide. For instance, 5xe2x80x2-ATGC and 5xe2x80x2-GCAT are complementary. The terms complement and complementary also encompass two polynucleotides where one polynucleotide contains at least one nucleotide that will not base pair to at least one nucleotide present on a second polynucleotide. For instance the third nucleotide of each of the two polynucleotides 5xe2x80x2-ATTGC and 5xe2x80x2-GCTAT will not base pair, but these two polynucleotides are complementary as defined herein. Typically two polynucleotides are complementary if they hybridize under certain conditions.
As used herein, xe2x80x9chybridizes,xe2x80x9d xe2x80x9chybridizing,xe2x80x9d and xe2x80x9chybridizationxe2x80x9d means that a single stranded polynucleotide forms a noncovalent interaction with a complementary polynucleotide under certain conditions, as described herein.
xe2x80x9cSupportxe2x80x9d as used herein refers to a matrix, for instance a filter, to which a microbe can be attached.
Unless otherwise specified, xe2x80x9ca,xe2x80x9d xe2x80x9can,xe2x80x9d xe2x80x9cthe,xe2x80x9d and xe2x80x9cat least onexe2x80x9d are used interchangeably and mean one or more than one.